The advent of DNA microarray technology makes it possible to build an array of hundreds of thousands of DNA sequences in a very small area, such as the size of a microscope slide. See, e.g., U.S. Pat. No. 6,375,903 and U.S. Pat. No. 5,143,854, each of which is hereby incorporated by reference in its entirety. The disclosure of U.S. Pat. No. 6,375,903, also incorporated by reference in its entirety, enables the construction of so-called maskless array synthesizer (MAS) instruments in which light is used to direct synthesis of the DNA sequences, the light direction being performed using a digital micromirror device (DMD). Using an MAS instrument, the selection of DNA sequences to be constructed in the microarray is under software control so that individually customized arrays can be built to order. In general, MAS based DNA microarray synthesis technology allows for the parallel synthesis of over 800,000 unique features each containing unique pre-selected oligonucleotides, in a very small area of on a standard microscope slide. The microarrays are generally synthesized by using light to direct which oligonucleotides are synthesized at specific locations on an array, these locations being called features.
With the availability of the entire genomes of hundreds of organisms, for which a reference sequence has generally been deposited into a public data base, microarrays have been used to perform sequence analysis on DNA isolated from such organisms. One technique that can be used to identify a genetic variant is to sequence the genomic DNA of an individual and then to compare that sequence to the reference sequence of that organism. It has been found that many differences in DNA sequence are presented as single variations in DNA sequence, often referred to as single nucleotide polymorphisms or SNPs. The sequence comparison between the test genome and the reference genome of a species has been generally by the brute force mechanism of capillary sequencing to identify the SNPs for that individual.
Specifically, another method of identifying genetic variations has been by resequencing (See Sakai et al., (1989) PNAS 86:6230-6234). One method of resequencing that has shown significant results utilizes oligonucleotide microarray technology (Hacia, et al., (1999) Nature Genetics, 21(1 Suppl):42-7.) In particular, this type of resequencing chip consists of a complete tiling of the reference sequence—that is, a chip containing one probe corresponding exactly to each 29-mer in the reference sequence—plus, for each base in this sequence, three mismatch probes: one representing each possible SNP at this position. In theory, any time a SNP is present, the mismatch probe representing this SNP will have a higher intensity signal than the corresponding probe that matches the reference sequence. However, due to unpredictability in signal strength, varying hybridization efficiency, and various other sources of noise, this method typically results in many base positions whose identities are incorrectly predicted. As such, alternative methods for efficiently and accurately resequencing DNA using microarrays to identify mutations in the genomes of organisms would be a desirable contribution to the art.